For this competing renewal project (R01HL63290-16), we propose to continue collaborative studies between the Zlokovic and Griffin labs aimed at advancing basic knowledge on protein C/activated protein C (APC) pathways in the central nervous system (CNS) with an overall goal of advancing from basic knowledge to translation to the clinic. Previous results funded by this R01 project led to discovery of direct vasculoprotective, blood-brain barrier (BBB)-stabilizing, neuroprotective and anti-inflammatory activities of APC and its cytoprotective-selective mutants using rodent models of stroke, brain trauma and chronic neuronal degeneration. This project identified APC receptors and downstream pathways in the CNS that mediate cytoprotective APC signaling in neurons, brain endothelium and at the BBB and showed that protease activated receptor-1 (PAR1) has a major role in protection. Our progress was translated in 2014 into clinical Phase II trial for ischemic stroke of 3K3A-APC, a 2nd generation cytoprotective APC. For extending this project, our goals include: 1) providing proof of concept for hypothesized mechanisms for neuroprotective activities of APC, 2) characterizing novel 3rd generation APC-mimetic agents, and 3) discovering improved biologics for ischemic stroke with potential for treating other neurologic diseases. Our exciting new findings and preliminary data show that activation of PAR1 by APC involves a novel cleavage of this receptor's N-terminal domain at Arg46 which reveals a novel tethered ligand ending at residue Asn47 that causes APC's biased, -arrestin-dependent cytoprotective signaling. In contrast, classical activation of PAR1 by thrombin involves cleavage at Arg41 resulting in G-protein signaling and cellular toxicity. APC can cleave PAR1 at both Arg41 and Arg46, and in vivo proof of concept is lacking for the attractive hypothesis that APC's neuroprotection is based on PAR1 cleavage at Arg46. We hypothesize that: i) APC cleavage of PAR1 at Arg46 protects CNS, BBB and neurons (AIM 1); ii) an APC-mimetic peptide with the sequence of PAR1 residues 47-66 (TR47) (i.e., the tethered agonist generated after Arg46 cleavage) elicits -arrestin 2-dependent cytoprotective signaling in brain endothelium and neurons in vitro and in vivo after ischemic stroke (AIM 2); and iii) APC mutants with enhanced ability to cleave PAR1 at Arg46 may provide 3rd generation neuroprotective biologics. PRELIMINARY DATA include: i) generation of new mouse lines carrying R41Q-PAR1 and R46Q-PAR1 to prevent cleavages at either Arg41 or Arg46; ii) proof for feasibility of studying the neuroprotective actions of the TR47 peptide; and iii) success at engineering novel APC variants with mutations that enhance PAR1 cleavage at Arg46. Neuroprotection will be assessed using in vitro models of BBB and neuronal injury, stroke models in mice and rats, magnetic resonance imaging, neuropathological analysis, confocal microscopy and behavioral testing. We expect that new knowledge generated from this project will likely have significant, direct impact for translation to the clinic for ischemic stroke and, by extension, implications for therapies for othr neurological disorders.